The goal of this research is to exploit techniques for the targeted manipulation of neural activity to identify, and functionally define, brain networks underlying specific behaviors. As a model for such investigations, we are identifying the networks that govern the behavioral program executed by adult fruit flies shortly after emergence from the pupal case. In two published reports from the past year (see Peabody et al., 2008 and Peabody et al., 2009), we demonstrated that this program consists of two principle phases, an adaptive behavioral phase, which mediates the search for a suitable environment, and an environmentally-insensitive phase, which drives expansion of the wings to make them flight-worthy. Focusing on the innate, environmentally-insensitive phase, we also showed that two anatomically and functionally distinct groups of neurons contribute to wing expansion. Work that we are now preparing for publication elucidates the functional roles of these groups and the key role played by the hormone bursicon. We have also initiated experiments to identify neurons responsible for the adaptive, environmentally-sensitive phase of behavior. Elucidation of the circuits underlying both phases of the post-emergence behavioral program promises a detailed understanding of how intrinsic and extrinsic factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Identification of the mechanisms by which neuronal networks interact and adapt to organize behavior should shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. Specific details of our accomplishments over the last year follow. As noted above, our efforts over the past year have focused on the mechanisms that govern the environmentally-insensitive phase of the post-emergence behavioral sequence of Drosophila. We have confirmed that this phase consists of two coordinately executed motor patterns and we have demonstrated that these patterns are regulated in an all-or-none fashion by the hormone bursicon. In addition, we have extended our earlier functional characterization of the bursicon-expressing neurons by applying a new tool that we developed for the targeted stimulation of neurons (i.e. UAS-TRPM8). This tool supplements tools that we have introduced previously for the constitutive silencing (i.e. UAS-EKO) and enhancement (i.e. UAS-NaChBac) of electrical activity in neurons and permits the acute activation of cells using small decrements in temperature. Using this tool, we have shown in recently published work (Peabody et al., 2009) that stimulation of a group of neurons that contains the bursicon-expressing neurons as a subset elicits the entire wing expansion program. In work that we are now preparing for publication, we show that the wing expansion program can be induced by stimulating the bursicon-expressing neurons alone, or, remarkably, by stimulating a single pair of these neurons located in the subesophageal ganglion (i.e. the BSEG). This finding was made possible by combining our new technique for neuronal activation with our previously developed Split Gal4 system for refined transgene targeting. The Split Gal4 system is a combinatorial method that relies on the independent targeting of the two component domains of the Gal4 transcription factor: the DNA-binding (DBD) and transcription activation (AD) domains. Each domain is fused to one of two complementary, heterodimerizing leucine zippers so that the DBD and AD domains associate in cells that express both. In these cells, and in these cells alone, transgenes downstream of Gal4s UAS binding site are expressed. We previously showed that the Split Gal4 system could be made more potent by using the AD of the HSV-1 VP16 transcription factor in place of the Gal4 AD, but only at the expense of infidelity in targeting. We have now rectified this problem by introducing a genetically optimized VP16 AD, the efficacy of which we demonstrated in work on visual processing in Drosophila carried out in collaboration with the laboratory of Chi-hon Lee (see Gao et al., 2008). In work related to wing expansion, we have exploited the Split Gal4 system by targeting the DBD domain to bursicon-expressing neurons (i.e. burs-DBD) and making enhancer trap lines that express the VP16 AD in arbitrary patterns that include different subsets of the bursicon-expressing neurons. During the last year, we completed screening of over 400 VP16 AD enhancer-trap lines and identified several that permit expression of UAS-transgenes in unique subsets of bursicon-expressing neurons when combined with the burs-DBD. Of greatest interest were lines that permitted exclusive targeting of the two anatomically distinct subgroups of bursicon-expressing neurons identified in our previous work. One subgroup was the BSEG;the other consists of 14 neurons in the abdominal ganglion (i.e. the BAG). By using the Split Gal4 system to specifically target UAS-TRPM8 to each of these two groups of neurons, we have shown that activation of the BSEG, but not the BAG, induces wing expansion. Consistent with previous suppression data, activation of only the BAG induces physiological changes in the wing, but fails to initiate the motor programs that drive expansion. The observation that BSEG stimulation initiates the behaviors that support wing expansion is consistent with the pattern of arborization of these neurons in the central nervous system, as described in Peabody et al. (2008). The fact that BSEG stimulation elicits the somatic, as well as the behavioral, components of wing expansion also places the BSEG neurons upstream of the BAG neurons in the regulation of this process and provides insight into the hierarchical organization of the wing expansion circuit. In general, investigation of the circuits that govern posteclosion behavior in Drosophila using the broad palette of tools we are developing should provide insight into the principles used by all nervous systems to generate and organize behavior. In addition, our work should serve as a proof of concept of a circuit mapping approach that can be extended to studies of mammalian behavior as similar tools become available for vertebrate organisms. Indeed, one of our goals is to extend those technologies that we find useful in the fly to mammalian model systems. To this end, we are currently collaborating with the Allen Institute for Brain Science to test the efficacy of the Split Gal4 technique in transgenic mice.